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1.
NPJ Breast Cancer ; 7(1): 140, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34707097

RESUMO

Reactivation of dormant cancer cells can lead to cancer relapse, metastasis, and patient death. Dormancy is a nonproliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and ZR-75-1, and proliferative D2A1 breast cancer cell line models in vivo and/or in vitro, and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro, we show that D2.OR and ZR-75-1 cells in response to a 3D environment or serum-free conditions are growth-arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy.

2.
Cell Death Dis ; 12(10): 932, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34642295

RESUMO

Glioblastoma (GBM) is a brain malignancy characterized by invasiveness to the surrounding brain tissue and by stem-like cells, which propagate the tumor and may also regulate invasiveness. During brain development, polarity proteins, such as Par3, regulate asymmetric cell division of neuro-glial progenitors and neurite motility. We, therefore, studied the role of the Par3 protein (encoded by PARD3) in GBM. GBM patient transcriptomic data and patient-derived culture analysis indicated diverse levels of expression of PARD3 across and independent from subtypes. Multiplex immunolocalization in GBM tumors identified Par3 protein enrichment in SOX2-, CD133-, and NESTIN-positive (stem-like) cells. Analysis of GBM cultures of the three subtypes (proneural, classical, mesenchymal), revealed decreased gliomasphere forming capacity and enhanced invasiveness upon silencing Par3. GBM cultures with suppressed Par3 showed low expression of stemness (SOX2 and NESTIN) but higher expression of differentiation (GFAP) genes. Moreover, Par3 silencing reduced the expression of a set of genes encoding mitochondrial enzymes that generate ATP. Accordingly, silencing Par3 reduced ATP production and concomitantly increased reactive oxygen species. The latter was required for the enhanced migration observed upon silencing of Par3 as anti-oxidants blocked the enhanced migration. These findings support the notion that Par3 exerts homeostatic redox control, which could limit the tumor cell-derived pool of oxygen radicals, and thereby the tumorigenicity of GBM.

3.
Metabolites ; 11(9)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34564442

RESUMO

The cytokine transforming growth factor-ß (TGF-ß) can induce normal breast epithelial cells to take on a mesenchymal phenotype, termed epithelial-to-mesenchymal transition (EMT). While the transcriptional and proteomic changes during TGF-ß-induced EMT have been described, the metabolic rewiring that occurs in epithelial cells undergoing EMT is not well understood. Here, we quantitively analyzed the TGF-ß-induced metabolic reprogramming during EMT of non-transformed NMuMG mouse mammary gland epithelial cells using nuclear magnetic resonance (NMR) spectroscopy. We found that TGF-ß elevates glycolytic and tricarboxylic acid (TCA)-cycle activity and increases glutaminolysis. Additionally, TGF-ß affects the hexosamine pathway, arginine-proline metabolism, the cellular redox state, and strongly affects choline metabolism during EMT. TGF-ß was found to induce phosphocholine production. A kinase inhibitor RSM-93A that inhibits choline kinase α (CHKα) mitigated TGF-ß-induced changes associated with EMT, i.e., increased filamentous (F)-actin stress fiber formation and N-Cadherin mesenchymal marker expression.

4.
J Pathol ; 255(3): 330-342, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34357595

RESUMO

Chondrocytes in mice developing osteoarthritis (OA) exhibit an aberrant response to the secreted cytokine transforming growth factor (TGF)-ß, consisting in a potentiation of intracellular signaling downstream of the transmembrane type I receptor kinase activin receptor-like kinase (ALK)1 against canonical TGF-ß receptor ALK5-mediated signaling. Unfortunately, the underlying mechanisms remain elusive. In order to identify novel druggable targets for OA, we aimed to investigate novel molecules regulating the ALK1/ALK5 balance in OA chondrocytes. We performed gene expression analysis of TGF-ß signaling modulators in joints from three different mouse models of OA and found an upregulated expression of the TGF-ß co-receptor Cripto (Tdgf1), which was validated in murine and human cartilage OA samples at the protein level. In vitro and ex vivo, elevated expression of Cripto favors the hypertrophic differentiation of chondrocytes, eventually contributing to tissue calcification. Furthermore, we found that Cripto participates in a TGF-ß-ALK1-Cripto receptor complex in the plasma membrane, thereby inducing catabolic SMAD1/5 signaling in chondrocytes. In conclusion, we demonstrate that Cripto is expressed in OA and plays a functional role promoting chondrocyte hypertrophy, thereby becoming a novel potential therapeutic target in OA, for which there is no efficient cure or validated biomarker. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

5.
J Cell Physiol ; 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34350982

RESUMO

The role of liver kinase B1 (LKB1) in glioblastoma (GBM) development remains poorly understood. LKB1 may regulate GBM cell metabolism and has been suggested to promote glioma invasiveness. After analyzing LKB1 expression in GBM patient mRNA databases and in tumor tissue via multiparametric immunohistochemistry, we observed that LKB1 was localized and enriched in GBM tumor cells that co-expressed SOX2 and NESTIN stemness markers. Thus, LKB1-specific immunohistochemistry can potentially reveal subpopulations of stem-like cells, advancing GBM patient molecular pathology. We further analyzed the functions of LKB1 in patient-derived GBM cultures under defined serum-free conditions. Silencing of endogenous LKB1 impaired 3D-gliomasphere frequency and promoted GBM cell invasion in vitro and in the zebrafish collagenous tail after extravasation of circulating GBM cells. Moreover, loss of LKB1 function revealed mitochondrial dysfunction resulting in decreased ATP levels. Treatment with the clinically used drug metformin impaired 3D-gliomasphere formation and enhanced cytotoxicity induced by temozolomide, the primary chemotherapeutic drug against GBM. The IC50 of temozolomide in the GBM cultures was significantly decreased in the presence of metformin. This combinatorial effect was further enhanced after LKB1 silencing, which at least partially, was due to increased apoptosis. The expression of genes involved in the maintenance of tumor stemness, such as growth factors and their receptors, including members of the platelet-derived growth factor (PDGF) family, was suppressed after LKB1 silencing. The defect in gliomasphere growth caused by LKB1 silencing was bypassed after supplementing the cells with exogenous PFDGF-BB. Our data support the parallel roles of LKB1 in maintaining mitochondrial homeostasis, 3D-gliomasphere survival, and hindering migration in GBM. Thus, the natural loss of, or pharmacological interference with LKB1 function, may be associated with benefits in patient survival but could result in tumor spread.

6.
Angiogenesis ; 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34379232

RESUMO

Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-ß (TGF-ß)/bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl-prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-ß/BMP signalling, endothelial cell dysfunction, and vascular remodelling in PAH is unknown. Here, we report that Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-ß signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-ß signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-ß/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH.

7.
Hepatology ; 2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34435364

RESUMO

BACKGROUND & AIMS: In patients with acute liver failure (ALF) who suffer from massive hepatocyte loss, liver progenitor cells (LPC) take over key hepatocyte functions, which ultimately determines survival. This study investigated how the expression of HNF4α, its regulators and targets in LPC determines clinical outcome of ALF patients. APPROACH & RESULTS: Clinicopathological associations were scrutinized in 19 ALF patients (9 recovered and 10 receiving liver transplantation). Regulatory mechanisms between follistatin, activin, HNF4α and coagulation factor expression in LPC were investigated in vitro and in metronidazole-treated zebrafish. A prospective clinical study followed up 186 cirrhotic patients for 80 months to observe the relevance of follistatin levels in prevalence and mortality of acute-on-chronic liver failure (ACLF). Recovered ALF patients robustly express HNF4α in either LPC or remaining hepatocytes. As in hepatocytes, HNF4α controls the expression of coagulation factors by binding to their promoters in LPC. HNF4α expression in LPC requires the FOXH1-SMAD2/3/4 transcription factor complex, which is promoted by the TGF-ß superfamily member activin. Activin signaling in LPC is negatively regulated by follistatin, a hepatocyte-derived hormone controlled by insulin and glucagon. In contrast to patients requiring liver transplantation, recovered patients demonstrate a normal activin/follistatin ratio, robust abundance of the activin effectors phosphorylated SMAD2 and HNF4α in LPC, leading to significantly improved coagulation function. A follow-up study indicated that serum follistatin levels could predict the incidence and mortality of ACLF. CONCLUSIONS: These results highlight a crucial role of the follistatin-controlled activin-HNF4α-coagulation axis in determining the clinical outcome of massive hepatocyte loss-induced ALF. The effects of insulin and glucagon on follistatin suggests a key role of the systemic metabolic state in ALF.

8.
Cancers (Basel) ; 13(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204675

RESUMO

Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.

9.
Trends Cell Biol ; 31(9): 705-707, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34330579

RESUMO

Editing linear polyubiquitination of protein substrates by LUBAC and OTULIN is known to play a critical role in immune responses. A recent study by Fu et al. reveals how reversible linear polyubiquitination of the activin receptor-like kinase (ALK1) controls developmental angiogenesis and how its dysfunction leads to vascular malformations in humans.

10.
Curr Protoc ; 1(5): e116, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33961349

RESUMO

The liver is a frequent site of cancer metastasis, but current treatment options for cancer patients with liver metastasis are limited, resulting in poor prognosis. Colonization of the liver by cancer cells is a multistep and temporally controlled process. Investigating this process in biological relevant settings in a dynamic manner may lead to new therapeutic avenues. Experimental mouse models of liver metastasis combined with high-resolution microscopy methods can facilitate study of the mechanisms that underlie the outgrowth of cancer cells in the liver. Intravital imaging can provide information on the behavior of tumor cells in their biological setting, in time frames of hours to days. In this unit, we describe the experimental induction of liver metastasis through administration of cancer cells into mice via mesenteric vein injection. The behavior of these injected cells can then be studied using intravital imaging by surgical exposure or through an abdominal imaging window. The approach is described for use with an upright multiphoton microscope, making it widely applicable. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Inducing liver metastasis through mesenteric vein injection Basic Protocol 2: Short-term imaging of tumor cells in mouse liver Basic Protocol 3: Long-term imaging of tumor cells in mouse liver using an abdominal imaging window.


Assuntos
Microscopia Intravital , Neoplasias Hepáticas , Animais , Diagnóstico por Imagem , Modelos Animais de Doenças , Humanos , Camundongos
11.
Biomedicines ; 9(2)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669809

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is an ultrarare congenital disease that progresses through intermittent episodes of bone formation at ectopic sites. FOP patients carry heterozygous gene point mutations in activin A receptor type I ACVR1, encoding the bone morphogenetic protein (BMP) type I serine/threonine kinase receptor ALK2, termed activin receptor-like kinase (ALK)2. The mutant ALK2 displays neofunctional responses to activin, a closely related BMP cytokine that normally inhibits regular bone formation. Moreover, the mutant ALK2 becomes hypersensitive to BMPs. Both these activities contribute to enhanced ALK2 signalling and endochondral bone formation in connective tissue. Being a receptor with an extracellular ligand-binding domain and intrinsic intracellular kinase activity, the mutant ALK2 is a druggable target. Although there is no approved cure for FOP yet, a number of clinical trials have been recently initiated, aiming to identify a safe and effective treatment for FOP. Among other targeted approaches, several repurposed drugs have shown promising results. In this review, we describe the molecular mechanisms underlying ALK2 mutation-induced aberrant signalling and ectopic bone formation. In addition, we recapitulate existing in vitro models to screen for novel compounds with a potential application in FOP. We summarize existing therapeutic alternatives and focus on repositioned drugs in FOP, at preclinical and clinical stages.

12.
J Vis Exp ; (168)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33720132

RESUMO

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-ß), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-ß-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-ß-induced EndMT. Using these techniques, we provide evidence that TGF-ß2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Endoteliais/metabolismo , Edição de Genes , Mesoderma/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Imunofluorescência , Camundongos , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genética
13.
Front Cell Dev Biol ; 9: 616610, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33644053

RESUMO

Endothelial-to-mesenchymal transition (EndMT) plays an important role in embryonic development and disease progression. Yet, how different members of the transforming growth factor-ß (TGF-ß) family regulate EndMT is not well understood. In the current study, we report that TGF-ß2, but not bone morphogenetic protein (BMP)9, triggers EndMT in murine endothelial MS-1 and 2H11 cells. TGF-ß2 strongly upregulates the transcription factor SNAIL, and the depletion of Snail is sufficient to abrogate TGF-ß2-triggered mesenchymal-like cell morphology acquisition and EndMT-related molecular changes. Although SLUG is not regulated by TGF-ß2, knocking out Slug also partly inhibits TGF-ß2-induced EndMT in 2H11 cells. Interestingly, in addition to SNAIL and SLUG, BMP9 stimulates inhibitor of DNA binding (ID) proteins. The suppression of Id1, Id2, or Id3 expression facilitated BMP9 in inducing EndMT and, in contrast, ectopic expression of ID1, ID2, or ID3 abrogated TGF-ß2-mediated EndMT. Altogether, our results show that SNAIL is critical and indispensable for TGF-ß2-mediated EndMT. Although SLUG is also involved in the EndMT process, it plays less of a crucial role in it. In contrast, ID proteins are essential for maintaining endothelial traits and repressing the function of SNAIL and SLUG during the EndMT process. These data suggest that the control over endothelial vs. mesenchymal cell states is determined, at least in part, by a balance between the expression of SNAIL/SLUG and ID proteins.

14.
Cell Stem Cell ; 28(4): 653-670.e11, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33561425

RESUMO

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR+ cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.


Assuntos
Medula Óssea , Transplante de Células-Tronco Hematopoéticas , Animais , Células da Medula Óssea , Células Endoteliais , Endotélio , Células-Tronco Hematopoéticas , Camundongos , Células Estromais
15.
Signal Transduct Target Ther ; 6(1): 8, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33414388

RESUMO

Transforming growth factor-ß (TGFß) family members are structurally and functionally related cytokines that have diverse effects on the regulation of cell fate during embryonic development and in the maintenance of adult tissue homeostasis. Dysregulation of TGFß family signaling can lead to a plethora of developmental disorders and diseases, including cancer, immune dysfunction, and fibrosis. In this review, we focus on TGFß, a well-characterized family member that has a dichotomous role in cancer progression, acting in early stages as a tumor suppressor and in late stages as a tumor promoter. The functions of TGFß are not limited to the regulation of proliferation, differentiation, apoptosis, epithelial-mesenchymal transition, and metastasis of cancer cells. Recent reports have related TGFß to effects on cells that are present in the tumor microenvironment through the stimulation of extracellular matrix deposition, promotion of angiogenesis, and suppression of the anti-tumor immune reaction. The pro-oncogenic roles of TGFß have attracted considerable attention because their intervention provides a therapeutic approach for cancer patients. However, the critical function of TGFß in maintaining tissue homeostasis makes targeting TGFß a challenge. Here, we review the pleiotropic functions of TGFß in cancer initiation and progression, summarize the recent clinical advancements regarding TGFß signaling interventions for cancer treatment, and discuss the remaining challenges and opportunities related to targeting this pathway. We provide a perspective on synergistic therapies that combine anti-TGFß therapy with cytotoxic chemotherapy, targeted therapy, radiotherapy, or immunotherapy.

16.
Clin Sci (Lond) ; 135(1): 35-52, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33399850

RESUMO

Cancers may escape elimination by the host immune system by rewiring the tumour microenvironment towards an immune suppressive state. Transforming growth factor-ß (TGF-ß) is a secreted multifunctional cytokine that strongly regulates the activity of immune cells while, in parallel, can promote malignant features such as cancer cell invasion and migration, angiogenesis, and the emergence of cancer-associated fibroblasts. TGF-ß is abundantly expressed in cancers and, most often, its abundance associated with poor clinical outcomes. Immunotherapeutic strategies, particularly T cell checkpoint blockade therapies, so far, only produce clinical benefit in a minority of cancer patients. The inhibition of TGF-ß activity is a promising approach to increase the efficacy of T cell checkpoint blockade therapies. In this review, we briefly outline the immunoregulatory functions of TGF-ß in physiological and malignant contexts. We then deliberate on how the therapeutic targeting of TGF-ß may lead to a broadened applicability and success of state-of-the-art immunotherapies.


Assuntos
Neoplasias/imunologia , Neoplasias/terapia , Fator de Crescimento Transformador beta/imunologia , Animais , Humanos , Inibidores de Checkpoint Imunológico , Imunidade Inata , Imunoterapia/métodos , Integrinas , Camundongos , Microambiente Tumoral
17.
Genome Biol ; 22(1): 54, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514403

RESUMO

BACKGROUND: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions. RESULTS: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on EnhancerTRAM2, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness. CONCLUSIONS: YAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis.

18.
Int J Mol Sci ; 22(2)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33418880

RESUMO

Transforming growth factor ß (TGFß) is a secreted growth and differentiation factor that influences vital cellular processes like proliferation, adhesion, motility, and apoptosis. Regulation of the TGFß signaling pathway is of key importance to maintain tissue homeostasis. Perturbation of this signaling pathway has been implicated in a plethora of diseases, including cancer. The effect of TGFß is dependent on cellular context, and TGFß can perform both anti- and pro-oncogenic roles. TGFß acts by binding to specific cell surface TGFß type I and type II transmembrane receptors that are endowed with serine/threonine kinase activity. Upon ligand-induced receptor phosphorylation, SMAD proteins and other intracellular effectors become activated and mediate biological responses. The levels, localization, and function of TGFß signaling mediators, regulators, and effectors are highly dynamic and regulated by a myriad of post-translational modifications. One such crucial modification is ubiquitination. The ubiquitin modification is also a mechanism by which crosstalk with other signaling pathways is achieved. Crucial effector components of the ubiquitination cascade include the very diverse family of E3 ubiquitin ligases. This review summarizes the diverse roles of E3 ligases that act on TGFß receptor and intracellular signaling components. E3 ligases regulate TGFß signaling both positively and negatively by regulating degradation of receptors and various signaling intermediates. We also highlight the function of E3 ligases in connection with TGFß's dual role during tumorigenesis. We conclude with a perspective on the emerging possibility of defining E3 ligases as drug targets and how they may be used to selectively target TGFß-induced pro-oncogenic responses.


Assuntos
Neoplasias/patologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Transição Epitelial-Mesenquimal , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica , Transdução de Sinais , Proteínas Smad/metabolismo , Ubiquitinação
19.
Protein Cell ; 12(2): 89-106, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32583064

RESUMO

Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions. Aberrant glycosylation can lead to uncontrolled cell proliferation, cell-matrix interactions, migration and differentiation, and has been shown to be involved in cancer and other diseases. The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream. This cellular transformation process, which is associated by morphological change, loss of epithelial traits and gain of mesenchymal markers, is triggered by the secreted cytokine transforming growth factor-ß (TGF-ß). TGF-ß bioactivity is carefully regulated, and its effects on cells are mediated by its receptors on the cell surface. In this review, we first provide a brief overview of major types of glycans, namely, N-glycans, O-glycans, glycosphingolipids and glycosaminoglycans that are involved in cancer progression. Thereafter, we summarize studies on how the glycosylation of TGF-ß signaling components regulates TGF-ß secretion, bioavailability and TGF-ß receptor function. Then, we review glycosylation changes associated with TGF-ß-induced epithelial-to-mesenchymal transition in cancer. Identifying and understanding the mechanisms by which glycosylation affects TGF-ß signaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.


Assuntos
Transição Epitelial-Mesenquimal , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Humanos
20.
Proc Natl Acad Sci U S A ; 117(47): 29684-29690, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33184177

RESUMO

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein-protein interaction involved in actin filament processing and cell migration.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Moléculas de Adesão Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Células Jurkat , Prolina/metabolismo , Ligação Proteica/efeitos dos fármacos , Peixe-Zebra
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